Mechanobiological Analysis of Nanoparticle Toxicity.

Nanoparticles (NPs) are commonly used in healthcare and nanotherapy, but their toxicity at high concentrations is well-known. Recent research has shown that NPs can also cause toxicity at low concentrations, disrupting various cellular functions and leading to altered mechanobiological behavior. While researchers have used different methods to investigate the effects of NPs on cells, including gene expression and cell adhesion assays, the use of mechanobiological tools in this context has been underutilized. This review emphasizes the importance of further exploring the mechanobiological effects of NPs, which could reveal valuable insights into the mechanisms behind NP toxicity. To investigate these effects, different methods, including the use of polydimethylsiloxane (PDMS) pillars to study cell motility, traction force production, and rigidity sensing contractions, have been employed. Understanding how NPs affect cell cytoskeletal functions through mechanobiology could have significant implications, such as developing innovative drug delivery systems and tissue engineering techniques, and could improve the safety of NPs for biomedical applications. In summary, this review highlights the significance of incorporating mechanobiology into the study of NP toxicity and demonstrates the potential of this interdisciplinary field to advance our knowledge and practical use of NPs.

Probing mechanobiological role of filamin A in migration and invasion of human U87 glioblastoma cells using submicron soft pillars.

Filamin A (FLNa) belongs to an actin-binding protein family in binding and cross-linking actin filaments into a three-dimensional structure. However, little attention has been given to its mechanobiological role in cancer cells. Here, we quantitatively investigated the role of FLNa by analyzing the following parameters in negative control (NC) and FLNa-knockdown (KD) U87 glioma cells using submicron pillars (900 nm diameter and 2 µm height): traction force (TF), rigidity sensing ability, cell aspect ratio, migration speed, and invasiveness. During the initial phase of cell adhesion (< 1 h), FLNa-KD cells polarized more slowly than did NC cells, which can be explained by the loss of rigidity sensing in FLNa-KD cells. The higher motility of FLNa-KD cells relative to NC cells can be explained by the high TF exerted by FLNa-KD cells when compared to NC cells, while the higher invasiveness of FLNa-KD cells relative to NC cells can be explained by a greater number of filopodia in FLNa-KD cells than in NC cells. Our results suggest that FLNa plays important roles in suppressing motility and invasiveness of U87 cells.

Decrease in membrane fluidity and traction force induced by silica-coated magnetic nanoparticles.

BACKGROUND: Nanoparticles are being increasingly used in biomedical applications owing to their unique physical and chemical properties and small size. However, their biophysical assessment and evaluation of side-effects remain challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. RESULTS: Overdose (1.0 µg/µL) treatment with MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. In addition, HEK293 cells were morphologically shrunk, and their aspect ratio was significantly decreased. We found that each traction force (measured in micropillar) was increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, the velocity of cell movement was also significantly decreased. Moreover, intracellular level of adenosine triphosphate (ATP) was also decreased in a dose-dependent manner upon treatment with MNPs@SiO2(RITC). To understand these biophysical changes in cells, we analysed the transcriptome and metabolic profiles and generated a metabotranscriptomics network, which revealed relationships among peroxidation of lipids, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipids and suppression of focal adhesion and cell movement. CONCLUSION: Taken together, our results demonstrated that overdose of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side-effects.

Effect of silica-coated magnetic nanoparticles on rigidity sensing of human embryonic kidney cells.

BACKGROUND: Nanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 µg/µL. These protruded structures are involved in a cell's rigidity sensing, but how these NPs affect rigidity sensing is unknown. RESULTS: Here, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 µg/µL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 µm in height, 24.21 nN/µm in stiffness k) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC). CONCLUSIONS: Our results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Silica-Coated Magnetic Nanoparticles Decrease Human Bone Marrow-Derived Mesenchymal Stem Cell Migratory Activity by Reducing Membrane Fluidity and Impairing Focal Adhesion.

For stem cell-based therapies, the fate and distribution of stem cells should be traced using non-invasive or histological methods and a nanomaterial-based labelling agent. However, evaluation of the biophysical effects and related biological functions of nanomaterials in stem cells remains challenging. Here, we aimed to investigate the biophysical effects of nanomaterials on stem cells, including those on membrane fluidity, using total internal reflection fluorescence microscopy, and traction force, using micropillars of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) labelled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)). Furthermore, to evaluate the biological functions related to these biophysical changes, we assessed the cell viability, reactive oxygen species (ROS) generation, intracellular cytoskeleton, and the migratory activity of MNPs@SiO2(RITC)-treated hBM-MSCs. Compared to that in the control, cell viability decreased by 10% and intracellular ROS increased by 2-fold due to the induction of 20% higher peroxidized lipid in hBM-MSCs treated with 1.0 µg/µL MNPs@SiO2(RITC). Membrane fluidity was reduced by MNPs@SiO2(RITC)-induced lipid oxidation in a concentration-dependent manner. In addition, cell shrinkage with abnormal formation of focal adhesions and ~30% decreased total traction force were observed in cells treated with 1.0 µg/µL MNPs@SiO2(RITC) without specific interaction between MNPs@SiO2(RITC) and cytoskeletal proteins. Furthermore, the migratory activity of hBM-MSCs, which was highly related to membrane fluidity and cytoskeletal abnormality, decreased significantly after MNPs@SiO2(RITC) treatment. These observations indicated that the migratory activity of hBM-MSCs was impaired by MNPs@SiO2(RITC) treatment due to changes in stem-cell biophysical properties and related biological functions, highlighting the important mechanisms via which nanoparticles impair migration of hBM-MSCs. Our findings indicate that nanoparticles used for stem cell trafficking or clinical applications should be labelled using optimal nanoparticle concentrations to preserve hBM-MSC migratory activity and ensure successful outcomes following stem cell localisation.